Therapeutic uses of BPI protein products in BPI-deficient humans

ABSTRACT

New therapeutic uses for BPI protein products that involve treatment of subjects with a BPI deficiency condition, including selective BPI deficiency, and newborns, including BPI-deficient newborns.

This application is a continuation-in-part of U.S. application Ser. No.09/285,124 filed Apr. 1, 1999, incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to novel therapeutic uses of BPIprotein products that involve treatment of BPI-deficient subjects.

BACKGROUND OF THE INVENTION

Newborns as a group are at increased risk for invasive bacterialinfections and resulting sepsis. Although the majority of theseinfections in newborns are caused by gram-positive organisms, a variablebut significant percentage of bacterial infections (about 20-40%) aredue to gram-negative bacteria, particularly E. coli, Haemophilusinfluenzae, Klebsiella spp., and Enterobacter spp. In fact, it is thegram-negative infections that are, in some studies, associated with thehighest mortality rate, which can be as high as about 40%. [Beck-Sague,C M et al., Pediatr Infect Dis J 13: 1110-116 (1994) and Stoll, B J etal., J Pediatr 129: 63-71 (1996)]

The mechanisms by which newborns are at increased risk for thesebacterial infections are not currently understood. Although theneutrophil defense system is innate, there are indications that itsfunction at birth is immature and suboptimal. Previous investigations ofthe activity of newborn neutrophils have demonstrated impairedadherence, chemotaxis, and phagocytosis. [Wright W C Jr. et al.,Pediatrics 56: 579-584 (1975); Cairo Miss., AJDC, 143:40-46 (1989);Schelonka R L et al., Sem. Perinatol., 22:2-14 (1998).] Impairedstimulus-induced adhesion and migration has been associated withdecreased surface expression of L-selectin and the β₂-integrin Mac-1.[Dinauer, M C, in “Hematology of Infancy & Childhood,” 5th ed. Nathanand Orkin, eds., Vol 1, pp 889-967 (1998).] These findings may explainthe difficulty in mobilizing neutrophils to sites of bacterial infectionbut do not explain the decreased phagocytic and bactericidal activity ofthe neutrophils of newborns.

Most studies of the microbicidal mechanism of newborn neutrophils havefocused on the oxidative mechanism (i.e., the phagocyteoxidase/MPO/hydroxyl radical system), with conflicting data indicatingeither increased or decreased capacity of this oxygen-dependentmechanism in newborns. [Dinauer, supra, and Ambruso et al., Ped Res18:1148-53 (1984).] Despite a growing literature on antibiotic proteinsand peptides, little is known about the oxygen-independent microbicidalmechanisms of newborn neutrophils. A slightly decreased content ofspecific (secondary) granules in the neutrophils of newborns has beendocumented, with an associated modest (≦2-fold) decrease in lysozyme andlactoferrin content relative to adult neutrophils. [Ambruso et al.,supra.] However, the major elements of the oxygen-independentantimicrobial arsenal of neutrophil primary granules, including BPI andthe defensin peptides, have not been assessed in neonates. Qing et al.,Infect. Immun., 64:4638-4642 (1996), compared the lipopolysaccharide(LPS) binding of newborn neutrophils to that of adult neutrophils andreported that the newborn neutrophils have lower levels ofmembrane-associated 55-57 kDa and 25 kDa proteins capable of bindingLPS. Although the missing proteins were not identified, the size andbinding properties of the 55-57 kDa protein appeared to be similar tothose of bactericidal/permeability-increasing protein (BPI) and thesurface LPS receptor CD14.

The rising tide of antibiotic resistance has placed renewed emphasis onthe development of agents to treat bacterial infection and its sequelae.Moreover, improved technology has led to increased survival rates forextremely ill full-term as well as premature neonates, which represent agrowing population at high risk for bacterial infection. Although thereplacement of neutrophils by granulocyte transfusion in newborns withsepsis has apparently been beneficial in some studies [Cairo et al.,Pediatrics 74: 887-92 (1984)] this potential therapy has beencomplicated by difficulty in obtaining histocompatible neutrophils andby transfusion reactions.

BPI is a protein isolated from the granules of mammalianpolymorphonuclear leukocytes (PMNs or neutrophils), which are bloodcells essential in the defense against invading microorganisms. HumanBPI protein has been isolated from PMNs by acid extraction combined witheither ion exchange chromatography [Elsbach, J. Biol. Chem., 254:11000(1979)] or E. coli affinity chromatography [Weiss, et al., Blood, 69:652(1987)]. BPI obtained in such a manner is referred to herein as naturalBPI and has been shown to have potent bactericidal activity against abroad spectrum of gram-negative bacteria. The molecular weight of humanBPI is approximately 55,000 daltons (55 kD). The amino acid sequence ofthe entire human BPI protein and the nucleic acid sequence of DNAencoding the protein have been reported in FIG. 1 of Gray et al., J.Biol. Chem., 264:9505 (1989), incorporated herein by reference. The Grayet al., nucleic acid and amino acid sequences are set out in SEQ ID NOS:1 and 2 hereto. U.S. Pat. Nos. 5,198,541 and 5,641,874 disclosesrecombinant genes encoding and methods for expression of BPI proteins,including BPI holoprotein and fragments of BPI. Recombinant human BPIholoprotein has also been produced in which valine at position 151 isspecified by GTG rather than GTC, residue 185 is glutamic acid(specified by GAG) rather than lysine (specified by AAG) and residue 417is alanine (specified by GCT) rather than valine (specified by GTT).

BPI is a strongly cationic protein. The N-terminal half of BPI accountsfor the high net positive charge; the C-terminal half of the moleculehas a net charge of −3. [Elsbach and Weiss (1981), supra.] A proteolyticN-terminal fragment of BPI having a molecular weight of about 25 kDpossesses essentially all the anti-bacterial efficacy of thenaturally-derived 55 kD human BPI holoprotein. [Ooi et al., J. Bio.Chem., 262: 14891-14894 (1987)]. In contrast to the N-terminal portion,the C-terminal region of the isolated human BPI protein displays someendotoxin-neutralizing activity and only slightly detectableanti-bacterial activity against gram-negative organisms. [Ooi et al., J.Exp. Med., 174:649 (1991).] An N-terminal BPI fragment of approximately23 kD, referred to as “rBPI₂₃,” has been produced by recombinant meansand also retains anti-bacterial activity against gram-negativeorganisms. [Gazzano-Santoro et al., Infect. Immun. 60:4754-4761 (1992).]An N-terminal analog of BPI, designated rBPI₂₁ [rBPI(1-193)ala¹³²], hasbeen produced as described in U.S. Pat. No. 5,420,019 and Horwitz etal., Protein Expression Purification. 8:28-40 (1996). An additionalN-terminal analog of BPI, designated rBPI(10-193)C132A orrBPI(10-193)ala¹³², has been produced as described in U.S. Pat. No.6,013,631.

The bactericidal effect of BPI was originally reported to be highlyspecific to gram-negative species, e.g., in Elsbach and Weiss,Inflammation: Basic Principles and Clinical Correlates, eds. Gallin etal., Chapter 30, Raven Press, Ltd. (1992). The precise mechanism bywhich BPI kills gram-negative bacteria is not yet completely elucidated,but it is believed that BPI must first bind to the surface of thebacteria through electrostatic and hydrophobic interactions between thecationic BPI protein and negatively charged sites on LPS. In susceptiblegram-negative bacteria, BPI binding is thought to disrupt LPS structure,leading to activation of bacterial enzymes that degrade phospholipidsand peptidoglycans, altering the permeability of the cell's outermembrane, and initiating events that ultimately lead to cell death.[Elsbach and Weiss (1992), supra]. LPS has been referred to as“endotoxin” because of the potent inflammatory response that itstimulates, i.e., the release of mediators by host inflammatory cellswhich may ultimately result in irreversible endotoxic shock. BPI bindsto lipid A, reported to be the most toxic and most biologically activecomponent of LPS.

BPI protein products have a wide variety of beneficial activities. BPIprotein products are bactericidal for gram-negative bacteria, asdescribed in U.S. Pat. Nos. 5,198,541, 5,641,874, 5,948,408, 5,980,897and 5,523,288. International Publication No. WO 94/20130 proposesmethods for treating subjects suffering from an infection (e.g.gastrointestinal) with a species from the gram-negative bacterial genusHelicobacter with BPI protein products. BPI protein products alsoenhance the effectiveness of antibiotic therapy in gram-negativebacterial infections, as described in U.S. Pat. Nos. 5,948,408,5,980,897 and 5,523,288 and International Publication Nos. WO 89/01486(PCT/US99/02700) and WO 95,08344 (PCT/US94/11255). BPI protein productsare also bactericidal for gram-positive bacteria and mycoplasma, andenhance the effectiveness of antibiotics in gram-positive bacterialinfections, as described in U.S. Pat. Nos. 5,578,572 and 5,783,561 andInternational Publication No. WO 95/19180 (PCT/US95/00656). BPI proteinproducts exhibit antifungal activity, and enhance the activity of otherantifungal agents, as described in U.S. Pat. No. 5,627,153 andInternational Publication No. WO 95/19179 (PCT/US95/00498), and furtheras described for BPI-derived peptides in U.S. Pat. No. 5,858,974, whichis in turn a continuation-in-part of U.S. application Ser. No.08/504,841 and corresponding International Publication Nos. WO 96/08509(PCT/US95/09262) and WO 97/04008 (PCT/US96/03845), as well as in U.S.Pat. Nos. 5,733,872, 5,763,567,5,652,332, 5,856,438 and correspondingInternational Publication Nos. WO 94/20532 (PCT/US/94/02465) and WO95/19372 (PCT/US94/10427). BPI protein products exhibit anti-protozoanactivity, as described in U.S. Pat. Nos. 5,646,114 and 6,013,629 andInternational Publication No. WO 96/01647 (PCT/US95/08624). BPI proteinproducts exhibit anti-chlamydial activity, as described in co-owned U.S.Pat. No. 5,888,973 and WO 98/06415 (PCT/US97/13810). Finally, BPIprotein products exhibit anti-mycobacterial activity, as described inco-owned, co-pending U.S. application Ser. No. 08/626,646, which is inturn a continuation of U.S. application Ser. No. 08/285,803, which is inturn a continuation-in-part of U.S. application Ser. No. 08/031,145 andcorresponding International Publication No. WO 94/20129(PCT/US94/02463).

The effects of BPI protein products in humans with endotoxin incirculation, including effects on TNF, IL-6 and endotoxin are describedin U.S. Pat. Nos. 5,643,875, 5,753,620 and 5,952,302 and correspondingInternational Publication No. WO 95/19784 (PCT/US95/01151).

BPI protein products are also useful for treatment of specific diseaseconditions, such as meningococcemia in humans (as described in U.S. Pat.Nos. 5,888,977 and 5,990,086 and International Publication No.WO97/42966 (PCT/US97/08016), hemorrhage due to trauma in humans. (asdescribed in U.S. Pat. Nos. 5,756,464 and 5,945,399. U.S. applicationSer. No. 08/862,785 and corresponding International Publication No. WO97/44056 (PCT/US97/08941), burn injury (as described in U.S. Pat. No.5,494,896 and corresponding International Publication No. WO 96/30037(PCT/US96/02349)) ischemia/reperfusion injury (as described in U.S. Pat.No. 5,578,568), and depressed RES/liver resection (as described inco-owned, co-pending U.S. application Ser. No. 08/582,230 which is inturn a continuation of U.S. application Ser. No. 08/318,357, which is inturn a continuation-in-part of U.S. application Ser. No. 08/132,510, andcorresponding International Publication No. WO 95/10297(PCT/US94/11404).

BPI protein products also neutralize the anticoagulant activity ofexogenous heparin, as described in U.S. Pat. No. 5,348,942, neutralizeheparin in vitro as described in U.S. Pat. No. 5,854,214, and are usefulfor treating chronic inflammatory diseases such as rheumatoid andreactive arthritis, for inhibiting endothelial cell proliferation, andfor inhibiting angiogenesis and for treating angiogenesis-associateddisorders including malignant tumors, ocular retinopathy andendometriosis, as described in U.S. Pat. Nos. 5,639,727, 5,807,818 and5,837,678 and International Publication No. WO 94/20128(PCT/US94/02401).

BPI protein products are also useful in antithrombotic methods, asdescribed in U.S. Pat. Nos. 5,741,779 and 5,935.930 and correspondingInternational Publication No. WO 97/42967 (PCT/US7/08017).

SUMMARY OF THE INVENTION

The present invention provides novel therapeutic uses for BPI proteinproducts that involve treatment of subjects, including humans, with aBPI deficiency condition, including a selective BPI deficiency. Anotheraspect of the invention provides treatment of newborns, includingBPI-deficient newborns, with BPI protein products. The invention isbased on the discovery that the neutrophils of newborns are selectivelydeficient in BPI, a protein that plays an important role in defendingagainst infection, including gram-negative bacterial infection.Treatment of subjects with a BPI deficiency condition is expected toalleviate adverse effects associated with this BPI deficiency,including, for example, decreasing susceptibility to infections,reducing the severity or invasiveness of the infection(s), andpreventing the sequelae of the infection(s).

It is contemplated that the administration of a BPI protein product to asubject may be accompanied by the concurrent administration of otherknown therapeutic agents appropriate for treating the subject.

Use of a BPI protein product in the manufacture of a medicament for thetreatment of humans with a BPI deficiency condition, including selectiveBPI deficiency, or a medicament for the treatment of newborns, includingBPI-deficient newborns, is also contemplated.

Numerous additional aspects and advantages of the invention will becomeapparent to those skilled in the art upon consideration of the followingdetailed description of the invention which describes presentlypreferred embodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 displays the relative BPI content of neonatal and adultneutrophils.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel therapeutic uses for BPI proteinproducts that involve treatment of subjects with a BPI deficiencycondition, including a selective BPI deficiency. Another aspect of theinvention provides treatment of newborns, including BPI-deficientnewborns, with BPI protein products. “Treatment” as used hereinencompasses both prophylactic and therapeutic treatment.

The invention is based on the discovery that neonatal neutrophils areselectively deficient in BPI. On average, the neonatal neutrophilscontained about 3-fold less BPI than the adult neutrophils, yet bothgroups contained nearly identical levels of other microbicidal proteins(e.g., MPO and defensin peptides) that are derived from the same primary(azurophil) granule compartment as BPI.

Although the average BPI content of newborn neutrophils wassignificantly lower than those of adults, it was not uniform. Somenewborns apparently contain larger BPI stores than others. About 40% ofnewborns were markedly deficient (˜9 to 10-fold less BPI than adults),with 33% of the neonatal samples showing no detectable levels of BPI atall. This variability suggests that BPI expression may be controlled byfactors that are not uniformly distributed in newborns and may explainwhy some newborns are at greater risk of gram-negative bacterialinfection than others.

The demonstration of such a BPI deficiency among newborns indicates thatsupplementation with BPI protein products may be of clinical benefit fornewborns, including premature newborns. Newborns constitute a patientpopulation that is at particularly high risk of infection and sepsiswith subsequent poor outcomes. This demonstration of a BPI deficientcondition, which has not previously been observed, also suggests thatnon-newborns, e.g., young children, older children or even adults, mayalso suffer from such a BPI deficiency and may benefit fromsupplementation with BPI protein products in amounts effective toalleviate the BPI deficiency. Such supplementation may provide aclinical benefit to such a BPI deficient subject. It is contemplatedthat supplementation is indicated whenever a BPI deficiency is observedor diagnosed, or whenever the subject is of a population with a highincidence of BPI deficiency (e.g., newborns), even if the subject is notsuffering from a condition associated with gram-negative bacteria andtheir endotoxin, for example, gram-negative bacterial infection,endotoxemia, or sepsis.

The invention thus contemplates methods for treating a subject with aBPI deficiency condition, including selective BPI deficiency, andmethods for treating newborns, including BPI-deficient newborns, whichcomprises administering an amount of a BPI protein product effective toalleviate the adverse effects of BPI deficiency. The treatment ofpremature and full-term neonates, whether healthy or suffering fromcongenital defects, illnesses, or infections, is contemplated.

The BPI protein product also may provide an added advantage of enhancingthe subject's resistance to or ability to fight infections, includinggram-negative bacterial, gram-positive bacterial and fungal infections,and prevention of the sequelae thereof. The administration of BPIprotein product is expected to reduce the incidence of severe orinvasive infection and to also reduce the incidence of adverse sequelaeof the infection. Such sequelae include, but are not limited to, asystemic inflammatory response, endotoxemia, bacterial and/orendotoxin-related shock and one or more conditions associated therewith,fever, tachycardia, tachypnea, cytokine overstimulation, increasedvascular permeability, hypotension, complement activation, disseminatedintravascular coagulation, anemia, thrombocytopenia, leukopenia,pulmonary edema, adult respiratory distress syndrome, intestinalischemia, renal insufficiency and failure, and metabolic acidosis.

“BPI-deficient newborn” means that the newborn's neutrophils containless BPI than the neutrophils of a normal adult. Correspondingly, a “BPIdeficiency condition” means a condition in which the amount of BPImeasured from the subject's neutrophils is less than the amount of BPImeasured from the neutrophils of a normal adult. Although the exactlevel of BPI for comparison purposes to determine a “deficient” levelwill depend on the quantitation technique used, an exemplary standardvalue is approximately 230 ng per 10⁶ neutrophils when a Western assayis used as described herein. Another exemplary standard value is 650 ngper 10⁶ neutrophils when a radioimmunoassay is used as described inWeiss and Olson, Blood, 69:652-659 (1987).

A subject with “selective BPI deficiency” means that the subject'sneutrophils contain less BPI than the neutrophils of a normal adult, yethave approximately normal levels of myeloperoxidase or defensins.

As used herein, “BPI protein product” includes naturally andrecombinantly produced BPI protein; natural, synthetic, and recombinantbiologically active polypeptide fragments of BPI protein; biologicallyactive polypeptide variants of BPI protein or fragments thereof,including hybrid fusion proteins and dimers; biologically activepolypeptide analogs of BPI protein or fragments or variants thereof,including cysteine-substituted analogs; and BPI-derived peptides. TheBPI protein products administered according to this invention may begenerated and/or isolated by any means known in the art. U.S. Pat. Nos.5,198,541 and 5,641,874, the disclosures of which are incorporatedherein by reference, disclose recombinant genes encoding, and methodsfor expression of, BPI proteins including recombinant BPI holoprotein,referred to as rBPI and recombinant fragments of BPI. U.S. Pat. No.5,439,807 and corresponding International Publication No. WO 93/23540(PCT/US93/04752), which are all incorporated herein by reference,disclose novel methods for the purification of recombinant BPI proteinproducts expressed in and secreted from genetically transformedmammalian host cells in culture and discloses how one may produce largequantities of recombinant BPI products suitable for incorporation intostable, homogeneous pharmaceutical preparations.

Biologically active fragments of BPI (BPI fragments) includebiologically active molecules that have the same or similar amino acidsequence as a natural human BPI holoprotein, except that the fragmentmolecule lacks amino-terminal amino acids, internal amino acids, and/orcarboxy-terminal amino acids of the holoprotein, including thosedescribed in U.S. Pat. Nos. 5,198,541 and 5,641,874. Nonlimitingexamples of such fragments include an N-terminal fragment of naturalhuman BPI of approximately 25 kD, described in Ooi et al., J. Exp. Med.174:649 (1991), and the recombinant expression product of DNA encodingN-terminal amino acids from 1 to about 193 to 199 of natural human BPI,described in Gazzano-Santoro et al., Infect. Immun. 60:4754-4761 (1992),and referred to as rBPI₂₃. In that publication, an expression vector wasused as a source of DNA encoding a recombinant expression product(rBPI₂₃) having the 31-residue signal sequence and the first 199 aminoacids of the N-terminus of the mature human BPI, as set out in FIG. 1 ofGray et al., supra, except that valine at position 151 is specified byGTG rather than GTC and residue 185 is glutamic acid (specified by GAG)rather than lysine (specified by AAG). Recombinant holoprotein (rBPI)has also been produced having the sequence (SEQ ID NOS: 1 and 2) set outin FIG. 1 of Gray et al., sierra, with the exceptions noted for rBPI₂₃and with the exception that residue 417 is alanine (specified by GCT)rather than valine (specified by GTT). Another fragment consisting ofresidues 10-193 of BPI has been described in U.S. Pat. No. 6,013,631,continuation-in-part U.S. application Ser. No. 09/336,402, filed Jun.18, 1999, and corresponding International Publication No. WO 99/66044(PCT/US99/13860), all of which are incorporated herein by reference.Other examples include dimeric forms of BPI fragments, as described inU.S. Pat. Nos. 5,447,913, 5,703,038, and 5,856,302 and correspondingInternational Publication No. WO 95/24209 (PCT/US95/03125), all of whichare incorporated herein by reference.

Biologically active variants of BPI (BPI variants) include but are notlimited to recombinant hybrid fusion proteins, comprising BPIholoprotein or biologically active fragment thereof and at least aportion of at least one other polypeptide, and dimeric forms of BPIvariants. Examples of such hybrid fusion proteins and dimeric forms aredescribed in U.S. Pat. No. 5,643,570 and corresponding InternationalPublication No. WO 93/23434 (PCT/US93/04754), which are all incorporatedherein by reference and include hybrid fusion proteins comprising, atthe amino-terminal end, a BPI protein or a biologically active fragmentthereof and, at the carboxy-terminal end, at least one constant domainof an immunoglobulin heavy chain or allelic variant thereof.

Biologically active analogs of BPI (BPI analogs) include but are notlimited to BPI protein products wherein one or more amino acid residueshave been replaced by a different amino acid. For example, U.S. Pat.Nos. 5,420,019, 5,674,834 and 5,827,816 and corresponding InternationalPublication No. WO 94/18323 (PCT/US94/01235), all of which areincorporated herein by reference discloses polypeptide analogs of BPIand BPI fragments wherein a cysteine residue is replaced by a differentamino acid. A stable BPI protein product described by this applicationis the expression product of DNA encoding from amino acid 1 toapproximately 193 or 199 of the N-terminal amino acids of BPIholoprotein, but wherein the cysteine at residue number 132 issubstituted with alanine and is designated rBPI₂₁Δcys or rBPI₂₁.Production of this N-terminal analog of BPI, rBPI₂₁, has been describedin Horvitz et al., Protein Expression Purification. 8:28-40 (1996).Similarly, an analog consisting of residues 10-193 of BPI in which thecysteine at position 132 is replaced with an alanine (designated“rBPI(10-193)C132A” or “rBPI(10-193)ala¹³²”) has been described in U.S.Pat. No. 6,013,631, continuation-in-part U.S. application Ser. No.09/336,402, filed Jun. 18, 1999, and corresponding InternationalPublication No. WO 99/66044 (PCT/US99/13860), all of which areincorporated herein by reference. Other examples include dimeric formsof BPI analogs; e.g. U.S. Pat. Nos. 5,447,913, 5,703,038, and 5,856,302and corresponding International Publication No. WO 95/24209(PCT/US95/03125), all of which are incorporated herein by reference.

Other BPI protein products useful according to the methods of theinvention are peptides derived from or based on BPI produced bysynthetic or recombinant means (BPI-derived peptides), such as thosedescribed in International Publication No. WO 97/04008 (PCT/US96/03845),which corresponds to U.S. application Ser. No. 08/621,259 filed Mar. 21,1996, and International Publication No. WO 96/08509 (PCT/US95/09262),which corresponds to U.S. Pat. No. 5,858,974, and InternationalPublication No. WO 95/19372 (PCT/US94/10427), which corresponds to U.S.Pat. Nos. 5,652,332 and 5,856,438, and International Publication No.WO94/20532 (PCT/US94/02465), which corresponds to U.S. Pat. No.5,763,567 which is a continuation of U.S. Pat. No. 5,733,872, which is acontinuation-in-part of U.S. application Ser. No. 08/183,222, filed Jan.14, 1994, which is a continuation-in-part of U.S. application Ser. No.08/093,202 filed Jul. 15, 1993 (corresponding to InternationalPublication No. WO 94/20128 (PCT/US94/02401)), which is acontinuation-in-part of U.S. Pat. No. 5,348,942, as well asInternational Application No. PCT/US97/05287, which corresponds to U.S.Pat. No. 5,851,802, the disclosures of all of which are incorporatedherein by reference. Methods of recombinant peptide production aredescribed in U.S. Pat. No. 5,851,802 and International Publication No.WO 97/35009 (PCT/US97/05287), the disclosures of which are incorporatedherein by reference.

Presently preferred BPI protein products include recombinantly-producedN-terminal analogs and fragments of BPI, especially those having amolecular weight of approximately between 20 to 25 kD such as rBPI₂₁ orrBPI₂₃, rBPI(10-193)C132A (rBPI(10-193)ala¹³²), dimeric forms of theseN-terminal proteins (e.g., rBPI₄₂ dimer), and BPI-derived peptides.

The administration of BPI protein products is preferably accomplishedwith a pharmaceutical composition comprising a BPI protein product and apharmaceutically acceptable diluent, adjuvant, or carrier. The BPIprotein product may be administered without or in conjunction with knownsurfactants or other therapeutic agents. A stable pharmaceuticalcomposition containing BPI protein products (e.g., rBPI₂₃) comprises theBPI protein product at a concentration of 1 mg/ml in citrate bufferedsaline (5 or 20 mM citrate, 150 mM NaCl, pH 5.0) comprising 0.1% byweight of poloxamer 188 (Pluronic F-68, BASF Wyandotte, Parsippany,N.J.) and 0.002% by weight of polysorbate 80 (Tween 80, ICI AmericasInc., Wilmington, Del.). Another stable pharmaceutical compositioncontaining BPI protein products (e.g., rBPI₂₁) comprises the BPI proteinproduct at a concentration of 2 mg/ml in 5 mM citrate, 150 mM NaCl, 0.2%poloxamer 188 and 0.002% polysorbate 80. Such preferred combinations aredescribed in U.S. Pat. Nos. 5,488,034, 5,696,090 and 5,955,427 andcorresponding International Publication No. WO 94/17819(PCT/US94/01239), the disclosures of all of which are incorporatedherein by reference. As described in U.S. Pat. No. 5,912,228 andcorresponding International Publication No. WO96/21436 (PCT/US96/01095),all of which are incorporated herein by reference, other poloxamerformulations of BPI protein products with enhanced activity may beutilized, optionally with EDTA.

Therapeutic compositions comprising BPI protein product may beadministered systemically or topically. Systemic routes ofadministration include oral, intravenous, intramuscular or subcutaneous,injection (including into a depot for long-term release), intraocularand retrobulbar, intraperitoneal, intraperitoneal (e.g. byintraperitoneal lavage), intrapulmonary (using powdered drug, or anaerosolized or nebulized drug solution), or transdermal.

When given parenterally, BPI protein product compositions are generallyinjected in doses ranging from 1 μg/kg to 100 mg/kg per day, preferablyat doses ranging from 0.1 mg/kg to 20 mg/kg per day, more preferably atdoses ranging from 1 to 20 mg/kg/day and most preferably at dosesranging from 2 to 10 mg/kg/day. The treatment may continue by continuousinfusion or intermittent injection or infusion, at the same, reduced orincreased dose per day for, e.g., 1 to 3 days, and additionally asdetermined by the treating physician. When administered intravenously,BPI protein products are preferably administered by an initial briefinfusion followed by a continuous infusion. The preferred intravenousregimen is a 1 to 20 mg/kg brief intravenous infusion of BPI proteinproduct followed by a continuous intravenous infusion at a dose of 1 to20 mg/kg/day, continuing for up to one week. A particularly preferredintravenous dosing regimen is a 1 to 4 mg/kg initial brief intravenousinfusion followed by a continuous intravenous infusion at a dose of 1 to4 mg/kg/day, continuing for up to 72 hours.

Topical routes include administration in the form of salves, creams,jellies, ophthalmic drops or ointments (as described in co-owned,co-pending U.S. application Ser. Nos. 08/557,289 and 08/557,287, bothfiled Nov. 14, 1995), ear drops, suppositories, irrigation fluids (for,e.g., irrigation of wounds) or medicated shampoos. For example, fortopical administration in drop form, about 10 to 200 μL of a BPI proteinproduct composition may be applied one or more times per day asdetermined by the treating physician.

Those skilled in the art can readily optimize effective dosages andadministration regimens for therapeutic compositions comprising BPIprotein product, as determined by good medical practice and the clinicalcondition of the individual subject.

“Concurrent administration,” or “co-administration,” as used hereinincludes administration of the agents, in conjunction or combination,together, or before or after each other. The BPI protein product andsecond agent(s) may be administered by different routes. For example,the BPI protein product may be administered intravenously while thesecond agent(s) is(are) administered intravenously, intramuscularly,subcutaneously, orally or intraperitoneally. The BPI protein product andsecond agent(s) may be given sequentially in the same intravenous lineor may be given in different intravenous lines. Alternatively, the BPIprotein product may be administered in a special form for gastricdelivery, while the second agent(s) is(are) administered, e.g., orally.The formulated BPI protein product and second agent(s) may beadministered simultaneously or sequentially, as long as they are givenin a manner sufficient to allow all agents to achieve effectiveconcentrations at the site of action.

Other aspects and advantages of the present invention will be understoodupon consideration of the following illustrative examples, which comparethe components of neutrophils from full term neonates and from adults,Example 1 addresses the relative BPI content of neonatal and adultneutrophils. Example 2 addresses the extracellular BPI levels ofneonatal and adult blood. Example 3 addresses the relative MPO anddefensin peptide content of neonatal and adult neutrophils. Example 4addresses effect of BPI protein product supplementation on antibacterialand cytokine-inducing activity of neonatal cord blood.

EXAMPLE 1 Comparison of BPI Content of Neonatal and Adult Neutrophils

In order to compare the BPI content of neonatal and adult neutrophils,cell-associated BPI was measured by Western blot analysis of neutrophildetergent extracts. The neutrophil content of BPI was then estimated byvisual comparison to two-fold dilutions of purified BPI, allowingquantitation of sample values.

Neonatal neutrophils were obtained from cord blood samples, which werecollected immediately after cesarean section or vaginal delivery. Cordblood was collected into sterile tubes anticoagulated with sodiumheparin (Becton Dickinson) and placed on ice. All samples were labelednumerically and the results kept anonymous. Adult neutrophils wereobtained from peripheral blood from healthy adult volunteers.

Neutrophils were isolated from whole blood as described in Le-v et al.,J. Immunol 154: 5403-10 (1995). Anticoagulated blood was promptly(within 30-60 minutes) processed by dextran sedimentation [3%pyrogen-free dextran (United States Biochemical) diluted in HanksBalanced Salt Solution without divalent cations, to avoid neutrophilclumping (Gibco BRL)]. Ficoll-hypaque (endotoxin-free Ficoll-Paque Plus,Pharmacia Biotech) gradient centrifugation was employed to generate aneutrophil-rich fraction. Brief hypotonic lysis (˜45 sec on ice) wasemployed to remove red blood cells. An automated total WBC count anddifferential (Technion H3 RTX automated cell counter, Miles) wasobtained on every sample prior to pelleting by centrifugation. Whitecell differential counts were often confirmed by Wright stain and manualassessment. Neutrophil viability was assessed by trypan blue exclusion.Neutrophil pellets (typically >85% pure) were frozen in Eppendorf tubesat −70° C. prior to batch analysis.

Western blots to determine relative BPI content were conducted asfollows. Neutrophils were thawed and solubilized with 4× SDS-PAGEloading buffer (0.8% SDS, 0.34 (v/v) glycerol, 0.04% Bromphenol Blue,0.02 M DTT, 0.2 Tris pH 6.8) prior to fractionation over a 10% SDS-PAGEgel (PAGE-ONE precast 10% gels, Owl Separation System). After Westerntransfer onto nitrocellulose (Protran BA85, pore size 0.45 μm,Schleicher & Schuell), and blocking of non-specific sites with 3% bovineserum albumin [BSA/Tris-buffered saline pH 7.4 (BTS)]. BPI was detectedusing 0.1% (v/v) whole anti-BPI goat serum as described in Levy et al.,J Clin Invest 94:672-682. (1994). Bound antibody was detected using: (a)0.05% (v/v) peroxidase-conjugated protein G followed by metal-enhanceddiaminobenzoic acid (DAB; Pierce), (b) 1:35,000 dilution ofperoxidase-conjugated protein G as part of the SuperSignalchemiluminescent system (Pierce), or (c) 0.1% (v/v) 1-125 protein G. Fordetection methods (b) and (c), signal was detected by exposing the blotsto Kodak XAR film. This Western transfer protocol provided detection inthe range of 10-200 nanograms with readily apparent differences insignal intensity between two-fold dilutions of a BPI standard, thusallowing interpolation of BPI content in test samples. Recombinant humanBPI (rBPI₅₀) was prepared as described in Horwitz et al., ProteinExpression & Purification 8: 28-40 (1996).

To compare analysis of BPI content by two independent techniques, anumber of neutrophil samples were extracted with sulfuric acid tosolubilize BPI as described in Levy et al., (1994), supra, and analyzedfor BPI content by both Western blotting and a sandwich ELISA assay asdescribed in White et al., J Immunol Methods 167:227-235 (1994). SimilarBPI levels were obtained by both techniques, indicating that the Westernblotting data are representative and relatively accurate.

Composite data from multiple Western blotting experiments are shown inFIG. 1, which is a scattergram of BPI content in the neutrophils ofnewborns and adults. Horizontal bars indicate average values fornewborns and adults. All samples of adult neutrophils (n=22, mean age 29years) contained quantifiable levels of BPI, the average of which was234+/−27 ng per 10⁶ neutrophils. In contrast, newborn neutrophils (n=21,mean gestational age 38.6 weeks) contained significantly lower amountsof BPI: 67+/−13 ng per 10⁶ neutrophils (p<0.001, 2-sided test). Medianvalues for BPI content of adult and newborn neutrophils were 200 ng and50 ng respectively. Thus, newborn neutrophils contained at least 3-foldless BPI than adult neutrophils.

It is also evident from FIG. 1 that about 40% (8 of 21) of the newbornneutrophils were markedly deficient in BPI. Among these eight samples,seven had no detectable BPI even after prolonged exposure. This numberrepresents 33% of the newborn patients studied. For the purposes ofquantitation, such samples were considered to contain one-half of thelowest amount of BPI that was detectable in the standard curve (i.e.about 10 ng per 10⁶ neutrophils). The analysis of BPI content was thusconservatively biased towards overestimating the amount of BPI innewborn neutrophils, with the actual difference in neutrophil BPIcontent of some newborns relative to adults perhaps being more than10-fold. Of note. MPO was easily detected in three newborns in whomthere was no detectable BPI.

EXAMPLE 2 Comparison of Extracellular Levels of BPI in Neonatal andAdult Plasma

To determine whether the relatively low BPI content of newbornneutrophils was related to degranulation, possibly secondary toperinatal stress, the levels of extracellular BPI in newborn plasmasamples and adult plasma samples were compared. Newborn and adult plasmasamples were collected within 30-60 minutes of drawing cord orperipheral venous blood, respectively. Samples were stored in cryogenicmicrotubes (Sarstedt) at −70° C. prior to batch analysis.

BPI content of plasma was determined employing a biotinylated anti-BPIantibody in a sandwich ELISA format as described in White et al. (1994),supra. This ELISA system yielded a linear range from 0.1 to 6 ng BPI/mland showed negligible cross reactivity with the homologouslipopolysaccharide-binding protein (LBP).

The average cord plasma BPI content was 16+/−3 ng/ml (n=13), which ishigher than that previously reported for plasma samples collected from20 healthy adults (<0.2 to 2.1 ng/ml; White et al., (1994), supra).However, calculated per cc of whole cord blood, this plasma content ofBPI represents less than 2% of cellular BPI content. Thus, there was noevidence for substantial extracellular degranulation of BPI at the timeimmediately preceding collection and processing of newborn cord blood.

EXAMPLE 3 Comparison of MPO and Defensin Levels in Neonatal and AdultNeutrophils

To assess whether other primary (azurophil) granule constituents werealso relatively decreased in newborn neutrophils, the content ofmyeloperoxidase (MPO) and of defensin peptides in neonatal and adultneutrophils was measured as follows.

Levels of myeloperoxidase (MPO) were detected by Western blotting using0.1% (v/v) rabbit anti-MPO serum [described in Nauseef et al., J ClinInvest 71: 1297-1307 (1983)] followed by 0.1% (v/v) I¹²⁵ protein G. Ascontrol for MPO blots, a two-fold dose curve of adult azurophil granulefraction (prepared as described in Borregaard et al., J Cell Biol 97:52-61 (1983) was solubilized in 4×SDS-PAGE buffer and analyzed as well.For purposes of quantitation, MPO content in neutrophil samples wasexpressed in “antigenic units” defined in relation to an adult azurophilgranule extract standard: one antigenic unit was set equal to the bandintensity of an azurophil granule extract sample representing 10⁶ adultneutrophil equivalents.

Levels of defensins were detected by subjecting neutrophil extracts fromadults and newborns to acid-urea (AU)-PAGE as described in Harwig etal., Meth Enzymol 236: 160-172 (1994). Briefly, neutrophils weresonicated in 5% acetic acid prior to overnight extraction at 4—C.Insoluble components were removed by centrifugation, supernatantslyophilized, and resuspended in AU-PAGE buffer prior to electrophoresisand Coomassie Brilliant Blue R stain. For each samples, the intensity ofstaining was visually compared to two-fold dilutions of controlextracts.

The results of Western blotting for MPO showed that the MPO content ofnewborn neutrophils (6.0+/−2.5 antigenic units per 10⁶ cells, n=7samples) and of adult neutrophils (4.3+/−1.6 antigenic units per 10⁶cells, n=7 samples) as not statistically different. Thus, in accordancewith previous observations by others (Kjeldsen et al., Pediatr Res 40:120-129 (1996), newborn and adult neutrophils appear to contain nearlyidentical amounts of MPO.

Despite the use of a sensitive detection technique which easily revealedtwo-fold differences in defensin content, the AU-PAGE results for thedefensin peptides showed that there was no discernible difference in thecontent of defensins in adult (n=8) and newborn (n=8) neutrophils. Ofnote, although the levels of lysozyme were somewhat decreased in some ofthe newborn neutrophil samples, the overall pattern of neutrophilproteins did not significantly, vary in migration or band intensitybetween newborn and adults.

Taken together, the results in Examples 1, 2 and 3 indicate that newbornneutrophils have intrinsically lesser quantities of BPI because: (a) apriori considerations would predict that BPI should remainintracellularly since it resides in the primary (azurophilic) granuleswhich are known to be the least easily mobilized compartment of bothadult and newborn neutrophils, (b) newborn and adult neutrophils containnearly identical amounts of both MPO and defensin, both of which arecomponents of the same primary (azurophil) granule where BPI is stored,and it is highly unlikely that selective degranulation of BPI occurred,and (c) levels of BPI in cord plasma represent only a small fraction(<2%) of total cellular BPI, suggesting that there was no significantrelease of BPI from cellular stores to the extracellular space at thetime immediately preceding cord blood collection.

EXAMPLE 4 Effect of BPI Protein Product Supplementation on AntibacterialActivity of Newborn Cord Blood

This experiment evaluated the effect of supplementation of exogenous BPIprotein product to newborn cord blood, as measured by effect on survivaland TNF-α cytokine-inducing activity of various gram-negative bacteria.Cord blood samples were collected immediately after vaginal delivery(n=17) or cesarean section (n=26) into sterile tubes anticoagulated withsodium citrate [sodium citrate (0.129M, 3.8%) tubes, Becton Dickinson(Franklin Lakes. NJ)].

The bacteria tested were E. coli K1/r, a K1-encapsulated, rough LPS,serum-resistant clinical isolate which has been shown to be sensitive toBPI-mediated killing both in artificial media and whole adult blood exvivo: Citrobacter koseri, isolated from the blood and cerebrospinalfluid of a 14 day old male with meningitis; and Klebsiella pneumoniae,Enterobacter agglomerans and Enterobacter cloaceae, as well as Serratiamarcescens isolated from blood cultures of newborns (7-27 day old) withcongenital cardiac defects requiring invasive surgery. Frozen stocks ofbacteria were prepared by culturing in trypticase soy broth [TSB, BectonDickinson & Co. Cockeysville, Md.], and adding sterile glycerol to 15%(vol/vol) prior to freezing at −80° C.

For bactericidal assays, subcultures of bacterial stocks were preparedby inoculating a loopful into 20 ml of TSB and incubating at 37 DC withshaking for about 4 hours (to late logarithmic phase growth). Bacterialconcentration was determined by measuring optical density (OD) at 550 nmin a spectrophotometer. Subcultures were harvested by centrifugation andresuspended in sterile physiologic saline to the desired concentration.Antibacterial assays were conducted in Eppendorf tubes [ResearchProducts International (Mount Prospect, Ill.)] in a total volume of 100μl. Samples contained 80 μl cord blood or buffered saline (20 mM sodiumphosphate pH 7.4/0.9% NaCl) as a control, 10 μl rBPI₂₁ (or bufferedsaline), and 10 μl of bacteria (added last, to a final concentration ofabout 10⁴/ml). Samples were incubated with shaking at 37° C. At 0, 30,90, and 180 minutes, 10 μl of each sample was plated on a Petri dish anddispersed with about 9 mL of molten (˜50° C.) Bactoagar containing 0.8%(wt/vol) nutrient broth [Difco Laboratories (Detroit, Mich.)] and 0.5%(wt/vol) NaCl. The agar was allowed to solidify at room temperature, andbacterial viability was measured as the number of colonies formed afterincubation of plates at 37° C. for 18 to 24 hours. Bacterial viabilitywas expressed as colony forming units (CFU) as a percentage of thebuffered saline control sample.

For cytokine-induction assays, bacteria were incubated in blood for 5hours to allow accumulation of TNF-α. Blood was diluted five-fold withRPMI [Gibco BRL, Grand Island, N.Y.] then centrifuged at 1000×g for 5min to collect the extracellular fluid (diluted plasma). Samples werestored frozen at 80° C. prior to measurement of TNF-α using a QuantikineTNF-α ELISA Kit [R&D System (Minneapolis, Minn.)] according to themanufacturer's instructions.

Results showed that while growth of E. coli K1/r was inhibited by adultblood, newborn cord blood served as a growth medium for this organismwhich grew logarithmically over several hours. Addition of rBPI₂₁ wasable to markedly diminish growth of this organism both in adult and newborn cord blood with an IC₅₀ of about 10 nM. Similarly, growth ofCitrobacter koserii was inhibited by adult blood but the organism grewlogarithmically in newborn cord blood. Addition of rBPI₂₁ provided areduction in bacterial growth with an IC₅₀ of about 1000 nM. K.pneumonziae, E. cloaceae and S. marcescens were relatively resistant toBPI protein product, while E. agglomerans was too rapidly killed by bothadult and newborn cord to observe an effect of BPI protein product.

Cytokine responses of newborn cord blood to E. coli K1/r were similar tothose of adult blood. Addition of rBPI₂₁ was able to inhibitbacteria-induced TNF-α release with similar potency in both adult andnewborn cord blood (IC₅₀ about 10-100 nM). C. koserii, K pneumoniae, E.cloaceae and agglomerans, and S. marcescens also induced substantialTNF-α release in both adult and newborn cord blood. The overall averageTNF-α release induced by all of the six gram-negative isolates testedwas closely similar in both adult and newborn cord blood. Addition ofrBPI₂₁ was able to inhibit induction of TNF-α release by all of thespecies tested (IC₅₀ ranging from about 1 to 1000 nM). C. koserii, about100 nM. K. pneumoniae about 10-100 nM, E. cloaceae about 100 nM, E.agglomerans about 10-100 nM and S. marcescens about 1-10 nM).

Thus, these results demonstrated that replenishment of BPI in the formof rBPI₂₁ enhanced the antibacterial activity of newborn cord bloodagainst E. coli K1/r and C. koserii and inhibited bacteria-inducedcytokine release.

Numerous modifications and variations of the above-described inventionare expected to occur to those of skill in the art. Accordingly, onlysuch limitations as appear in the appended claims should be placedthereon.

1. A method of treating a subject with a BPI deficiency conditioncomprising administering to said subject an amount of BPI proteinproduct effective to alleviate the BPI deficiency.
 2. The method ofclaim 1 wherein the subject is an adult.
 3. The method of claim 1wherein the condition is a selective BPI deficiency.
 4. The method ofclaim 1 wherein the subject is a newborn.
 5. The method of claim 4wherein the newborn is a premature newborn.
 6. The method of claim 1wherein the BPI protein product is rBPI₂₁.
 7. The method of claim 1wherein the BPI protein product is an N-terminal fragment of BPI havinga molecular weight approximately between about 20 to 25 kd.
 8. Themethod of claim 1 wherein the BPI protein product is rBPI(10-193)ala¹³².9. The method of claim 1 wherein the BPI protein product is rBPI₅₀.